Method for preparing an Eruca sativa extract and use for shaving

ABSTRACT

The invention is directed to an aqueous extract of  Eruca sativa  (arugula) leaves that has antimicrobial activity on Gram-positive bacteria and mycoplasmas. The extract may be purified away from solid or insoluble components, standardized based on the weight of its non-aqueous or solid content, assayed for antimicrobial activity, and provided in an aseptic or sterile form for pharmaceutical use. It may be used to kill or inhibit the growth of microorganisms such as Gram positive bacteria, promote wound healing, or as prophylaxis against host colonization or infection by a microorganism.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/149,674, now allowed, having a filing date of Oct. 2, 2018.

BACKGROUND OF THE INVENTION Field of the Invention

The invention falls within the fields of biochemistry and microbiology.It pertains to an aqueous extract of leaves of Eruca sativa sometimesknown as arugula that can be used to inhibit the growth of Gram-positivebacteria such as those that contaminate skin wounds and lesions. Erucasativa is an edible plant within the Brassicaceae family.

Description of Related Art

This “background” description provides a general context helpful inunderstanding the invention. The work of the presently named inventor(s)to the extent that it is described in this section, as well as aspectsof the description which may not otherwise qualify as prior art at thetime of filing, are neither expressly or impliedly admitted as prior artagainst the present invention.

Plant extracts have been used in traditional medicine since ancienttimes to treat or eliminate disease. Building on this foundation oftraditional medicine, an impressive number of drugs have been isolatedfrom plant sources for treatment of human, animal and plant diseases.The new herbal extracts and drugs derived from them are used to treatdiseases or infestations caused by viruses, bacterial, fungi, parasitesand insects. Modern biochemists, microbiologists and physicians continuethe quest to discover new plant extracts and components useful inagronomy, animal science and medicine.

Phytochemicals and extracts from the Brassicaceae family contributebeneficial components to the human diet and are being studied todetermine whether they contain other biologically beneficial components.WO2006136933A2 and WO1993005153A1 involve evaluation of extracts ofBrassicaceae seeds for use as a pesticide or as an antifungal orantibacterial agent. US 2008/0182751 A1 evaluated processed plantmaterial from Brassicaceae for use against plant pests. WO2011099878A2evaluated antimicrobial effects of a fermented concoction of tropicalplants including those of Brassicaceae. FR2950250A1 describes organicsolvent extracts of Brassicaceae. Koubaa, et al., Free Radicals andAntioxidants 5(10): 29-34 (2015) evaluated the antibacterial activity ofnon-polar compounds extracted by hexane from Eruca sativa flowers.Alaraidh, et al., Iran J. Biotech. 12(1) (2014) describes antimicrobialsilver nanoparticles produced using Eruca sativa leaf extracts. Prasad,Int. J. Pure and Appl. Biosci. 2(2):158-162 (2014) evaluatedantimicrobial properties of organic solvent extracts of plants in theBrassica oleracea family. Despite much interest and ongoing research,much is still not known about which plants, or plant extracts, containbeneficial components.

In view of the emerging antibiotic resistance of many Gram positivebacteria, such as methicillin-resistant staphylococcus (MRSA), there isa significant need and demand for new antimicrobial drugs and agentsespecially those derived from non-toxic natural components such as Erucasativa. With this objective in mind the inventors diligently sought toidentify fractions and components of Eruca sativa that exhibitantimicrobial activity.

BRIEF DESCRIPTION OF THE INVENTION

An object of the invention is to provide therapeutic or antimicrobialcompositions obtained from aqueous extraction of Eruca sativa leaves andmethods for extracting and purifying these compositions. Advantageously,in some embodiments the leaf extract can be purified away from solid orinsoluble components of Eruca sativa, standardized based on the weightof its anhydrous or solid content, assayed for antimicrobial activity,and provided in an aseptic or sterile form for therapeutic use.

A further object of the invention is to provide methods for treatinginfections caused by Gram-positive bacteria and mycoplasmas using thesetherapeutic compositions.

An additional object of the invention is to provide methods forpromoting wound healing using these therapeutic compositions.

A further object of the invention is to provide methods for preventingthe growth or colonization of a subject or object with Gram-positivebacteria or mycoplasmas using antimicrobial compositions of theinvention.

These and other objects of the invention will become evident from thisdisclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B depict Eruca sativa and its leaves.

DETAILED DESCRIPTION OF THE INVENTION

The invention generally pertains to an aqueous extract of the leaves ofEruca sativa, compositions incorporating this extract, and methods forusing this extract to inhibit the growth of Gram-positive bacteria,mycoplasma and other microorganisms, or as prophylaxis against infectionor colonization of a host or object with such microorganisms.

Compositions containing these extracts can be administered to treat orpromote the healing of injuries including those to the skin, hair andnails (including pimples, acne, and sites of dermatitis, and dermaldamage associated with aging), to mucous membranes such as those to theeyes, the nasal and oral cavities, urinary tract, reproductive organs,GI tract, or to other tissues susceptible to microbial colonization orinfection.

The invention is described by reference to the following definitions andfeatures. Various embodiments of the invention may incorporate one ormore features, elements, ranges or alternatives described below.

An “Eruca sativa” extract contains one or more components besides wateraqueously extracted from the leaves of Eruca sativa, sometimes known asarugula, rocket salad, rucuola, rucoli, rugla, colewort or roquette. Insome embodiments, the extract is made from fresh leaves, in others fromdried, frozen or otherwise preserved leaves.

An aqueous extract is prepared by mashing, macerating, blending,sonicating, freeze-thawing, French pressing, digesting, or otherwisedisrupting Eruca sativa leaves so that water-soluble components can beremoved and recovered. Generally extraction is performed in an aqueousor water-based solution which may contain salt(s), buffers, stabilizers,enzymes, chelators (e.g., of divalent cations or metals like iron),antioxidants and/or preservatives. In some embodiments water-solublecomponents from Eruca sativa leaves are extracted using non-water polarsolvents or solvents that are miscible with water, or mixtures of thesesolvents with water.

A solution for aqueous extraction of leaves will contain sufficientwater or other aqueous solvent to extract components of Eruca sativathat are soluble in water. Such a solvent may contain 0 to 100 wt %water or any endpoint or intermediate value within this range, forexample, 100, 95, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25,20, 15, 10, or 5 wt % water. The extraction may be made using a singleaqueous phase or may be made using a multiple phase extraction, such aswith an aqueous solvent in combination with one or more immisciblephases; e.g., an extraction in which hydrophilic solutes go into anaqueous phase and hydrophobic solutes go into a hydrophobic phase.

In some embodiments water-soluble components of Eruca sativa leaves areextracted by steam, water vapor or another gaseous or vaporous hydrophilor polar solvent.

Polar solvents include those having a dielectric constant of at least15, 20, 30, 40, 50, 60, 80 or more or any intermediate value within thisrange. Examples of polar solvents include water, methanol, ethanol,n-propanol, isopropanol, DMSO, and mixtures thereof.

In other embodiments extraction may be performed under acidic, neutralor alkaline conditions ranging from pH 1.0 to pH 14.0 and allintermediate subranges and values. These include extraction at a pH of2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or9.0. In one embodiment, extraction is performed at a pH ranging from 6.0to 8.0.

In some embodiments, extraction times range from <5, 10, 15, 30, or 60minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, 24 ormore hours.

To facilitate the extraction of soluble components surfactants,chelators, and/or enzymes that digest Eruca sativa leaves, such ascellulase (e.g., endo-1,4-beta-D-glucanase (beta-1,4-glucanase,beta-1,4-endoglucan hydrolase, endoglucanase D,1,4-(1,3,1,4)-beta-D-glucan 4-glucanohydrolase), carboxymethyl cellulase(CMCase), avicelase, celludextrinase, cellulase A, cellulosin AP, alkalicellulase, cellulase A 3, 9.5 cellulase, and pancellase SS), proteases(e.g., serine-, cysteine-, threonine-, aspartic-, glutamic-, ormetallo-proteases, and asparagine peptide lyases), or nucleases (RNAase,DNAase), may be used.

Extraction may be performed at different temperatures, for example, from0, 5, 10, 15, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, or 100° C. (or at higher temperatures under pressure) or anyintermediate temperature within this range.

An Eruca sativa leaf extract can be further purified by removinginsoluble or solid components from an aqueous extract, for example, byseparating these solid components from liquid components bycentrifugation or filtration. Protein and non-protein components may beseparated or isolated from one another by filtration, precipitation,chromatography or by addition of a protease.

Proteins may be isolated by the chromatographic procedures describedherein or by precipitation, and/or differential solubilization.

Oils and hydrophobic components removed by aqueous extraction may beseparated from aqueous components by phase partition based on theirdifferential solubility in non-aqueous solvents, such as a solventhaving a dielectric constant of less than 15, 10, 5, or 2. Nonpolarsolvents include chloroform, diethylether, hexanes, benzene and toluene

An extract can be dialyzed to remove salts or other cations and anionsor other low molecular weight solutes. Dialysis membranes havingmolecular weight cutoffs ranging from 1, 10, 50, 100, 500, 1,000, 5,000,10,000, 50,000, 100,000, 500,000 or 1,000,000 kDa or any endpoint orintermediate cutoff value within this range may be employed for thispurpose.

A dialysis buffer can be selected to effect solvent exchange in anextract, for example, to alter the ionic concentration or pH of anextract.

One skilled in the art can select an appropriate dialysis buffer,temperature, and duration, for example, dialysis may be performed at 4°C. or at 25° C. for 2, 4, 6, 8, 10, 12, 18, or 24 hours. Dialysis buffermay be replaced and dialysis repeated 1, 2 or more times if desired.

An aqueous extract may be further purified chromatographically. Forexample, an aqueous extract may be fractionated by size exclusionchromatography, affinity chromatography, HPLC, hydrophobic interactionchromatography, ion-exchange chromatography or free-flow electrophoresisby methods known to those skilled in the art.

An Eruca sativa extract may be further treated to render it stable,aseptic or sterile. Such methods include but are not limited tofiltration, heat pasteurization or sterilization, addition of chelatorssuch as EDTA, EGTA, vitamin C or other antioxidants, or oxidants such ashydrogen peroxide. Microbial contaminants may be removed by filtration,for example, through a 0.1, 0.2, 0.22, or 0.45 micron filter. A freshextract may be refrigerated, frozen, or lyophilized for storage.

A purified Eruca sativa aqueous extract, preferably one having the solidleaf components removed, can be concentrated by removing a portion of orall of the aqueous solvent. A concentrated aqueous extract will have alower concentration of water and higher relative concentration ofwater-soluble components from Eruca sativa than an original or startingextract. A concentrated extract may contain 1, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 95 or <100 wt % of water or the aqueous solventcontained in the original leaf extract. This range includes anyintermediate value.

An anhydrous, dry or desiccated Eruca sativa extract will havesubstantially all of the aqueous solvent removed from it.

Concentration and dehydration methods are known in the art and include,but are not limited to, those performed with a device such as hot airblower, dryer, cylindrical dryer, zeolite dryer, a desiccator, or afreeze-dryer. An extract can be concentrated or dried by evaporating thesolvent using a falling film device, low pressure evaporator, or spraytower.

A fresh, concentrated, or dried extract may be stored under sterile oraseptic conditions that prevent loss of its antimicrobial activity, forexample, it may be stored in liquid nitrogen, or at −86° C., −70° C.,−20° C., 4° C. or stored in desiccated form in sealed ampules.

Eruca sativa compositions. The term “composition” includes those withone or more ingredients. Thus, an original aqueous extract of Erucasativa without further additions is a composition. A composition maycontain fewer ingredients than those present in an original aqueousextract provided that a therapeutic or antimicrobial activity isretained. For example, it can omit water or salts present in an originalaqueous extract or consist of the anhydrous components of a leafextract.

In some embodiments, a composition will contain one or more ingredients,one or more combinations of ingredients, or a greater content of aningredient, than those or that present in the original aqueous extract.For example, it may contain a preservative, antioxidant, chelator, or apharmaceutically acceptable carrier or excipient not present in anoriginal Eruca sativa aqueous extract or may be a combination of anEruca sativa extract and an aqueous or non-aqueous extract of anotherplant.

A composition may contain 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0,7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 99 or 100 wt % (or any endpoint or intermediate valuewithin this range) of an Eruca sativa extract, for example, it maycontain 1 wt % of an anhydrous Eruca sativa extract and 99 wt % water orother carrier or excipient. Preferably, it will contain atherapeutically effective amount of the extract. By an “effective”amount or a “therapeutically effective” amount of the Eruca sativaextract is meant a nontoxic but sufficient amount of the agent toprovide a beneficial effect. The amount of active agent that is“effective” will vary from subject to subject, depending on the age andgeneral condition of the individual, the particular active agent oragents, and the like. Unless otherwise indicated, the term“therapeutically effective” amount as used herein is intended toencompass an amount effective for the prevention, the amelioration of anadverse condition and/or treatment of the adverse condition.Determination of a therapeutic effect may be made by comparison with anotherwise identical composition that does not contain the Eruca sativaextract, or which contains a control extract from a different plant.

The terms “treating” and “treatment” as used herein refer to theadministration of a composition containing an Eruca sativa extract to aclinically symptomatic individual afflicted with an adverse condition,disorder, or disease, so as to effect a reduction in severity and/orfrequency of symptoms, eliminate the symptoms and/or their underlyingcause, and/or facilitate improvement or remediation of damage.Analogously, this term also applies to treatment of objects, such ascontacting an extract with a surface or surgical equipment to kill,inhibit or remove microbial contaminants.

The terms “preventing” and “prevention” refer to the administration ofan agent or composition to a clinically asymptomatic individual who issusceptible to a particular adverse condition, disorder, or disease, andthus relates to the prevention of the occurrence of symptoms and/ortheir underlying cause. For example, treatment may decrease in thenumber of Gram-positive bacteria in a wound, reduce inflammation,accelerate wound healing, or prevent or ameliorate scar formation.Analogously, this term also applies to prevention of bacterialcontamination of objects, such as contacting an extract with a surfaceor surgical equipment to prevent or remove microbial contamination.

Therapeutic Compositions. A composition may be formulated fortherapeutic use, for example, with a pharmaceutically acceptableexcipient or excipient in aseptic or sterile form. It may also includeother therapeutically active ingredients, such as anesthetics, plantextracts, antibiotics, or other antimicrobial agents.

In some embodiments, the composition of the invention will incorporateone or more non-Eruca sativa antimicrobial or antiviral herbal extracts.Such herbs include Calendula officinalis, Cinnamomum zeylanicum,Syzygium aromaticum, Allium sativum, Echinacea angustifolia, Mahoniaaquifolium, Althaea officinalis L.), Usnea barbata, Arctostaphylosuva-ursi, Achillea millefolium, Astragalus membranaceus, Uncariatomentosa, Vaccinium macrocarpon, Sambucus nigra, Zingiber officinale,Melissa officinalis, Glycyrrhiza glabra, Verbascum Thapsus, Oleaeuropaea, and/or Origanum vulgare. These herbal extracts include the oiland oily or dry components of these herbs as well as their aqueouscomponents. An appropriate content of Eruca sativa and one or more otherherbal extracts may be selected based on maximizing the antimicrobialactivity of the mixtures. In some embodiments the ratio of Eruca sativaaqueous extract to another herbal extract will range from 0.1 to 100 to100 to 0.1 as well as any endpoint or intermediate ratio. Such ratiosinclude 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:20, and 1:50and may be based on the weight on anhydrous or solid components of theextracts.

In other embodiments, extracts from cruciferous vegetables or chemicalcomponents of cruciferous vegetables may be added or incorporated intoan Eruca sativa extract. Cruciferous vegetables act as good sources ofnatural compounds because of high levels of carotenoids, tocopherols,and ascorbic acid.

In some embodiments, an Eruca sativa extract may be supplemented withnatural chemical compounds present in Eruca sativa. E. sativa has beenrecognized as a rich source of health-promoting phytochemical s,vitamins, carotenoids, fibers, minerals, glucosinolates,isothiocyanates, flavonoids such as kaempferol, quercetin, andisorhamnetin, flavanols, and phenolic compounds. Glucosinolates can beused to increase or confer anticarcinogenic, antifungal, antibacterial,and antioxidant activities on an extract. Addition of flavonoids mayincrease or confer additional anti-inflammatory, estrogenic, enzymeinhibition, antimicrobial, antiallergic, vascular, cytotoxic, antitumor,antioxidant, and free radical scavenging activities on an Eruca sativaextract.

The Eruca sativa leaf extract of the invention may also be formulatedalong with, or used or administered in conjunction with, traditionalremedies prepared from alum, anise, arak, asafetida, banana, black seed(Nigella sativa), caraway, cardamom, chamomile, cucumber, frankincense,garlic, myrrh, nakhwa, petroleum (naft, batrul), pomegranate, saffron,thyme, turmeric, or walnut bark. Treatments and modes of administrationare incorporated by reference to hypertext transferprotocol://archive.aramcoworld.com/issue/200605/natural.remedies.of.arabia.htm(last accessed Apr. 13, 2017). Plant extracts may be in the form of anaqueous, alcohol or other organic solvent, or oily extract, may be indry or powdered form, or may contain purified active components fromthese traditional remedies.

In some embodiments a composition of the invention will contain anantimicrobial component such as those described by Capelli, U.S. Pat.No. 5,662,913, Kaiser, et al., U.S. Pat. No. 6,383,505 B1, Mansouri,U.S. Pat. No. 6,579,516, and Koller, et al., U.S. Pat. No. 8,980,243,each of which is incorporated by reference.

Advantageously a composition will contain a concentration of Erucasativa extract and/or other active ingredients, sufficient to prevent orinhibit the growth of a target microorganism such as the Gram positivebacteria described herein. When a composition includes both the Erucasativa extract and another active ingredient it may exhibit additive orsynergistic therapeutic and/or antimicrobial activity, or the mixture oftwo active ingredients may expand its antimicrobial spectrum.

In some embodiments, the therapeutic or antimicrobial Eruca sativa-basedcompositions will be formulated for application to wounds in skin ormucous membranes, such as epidermal wounds or wounds in the eyes, noseor the mouth. For example, in the treatment of eye infections or otherinfections of mucous membranes, the composition, in the form of asolution, wash, lotion, emulsion, ointment or a cream, can be applied tothe mucous membrane of the patient using standard techniques. Standardmethods known in the medical and pharmaceutical arts are used toformulate and apply these compositions to wounds.

In the treatment of mouth infections, including gingivitis, thecomposition may be formulated as a solution or cream can be appliedusing a sponge applicator or a toothbrush. It may also be incorporatedinto a gargling solution, mouthwash or rinse.

The compositions of the invention may also be in the form of a solutionand used for infusing into a body cavity (e.g., a surgical wash) andthereby treating or reducing the risk of infection or may be prepared inan inhalable or aerosol form for administration to the respiratorysystem.

In other embodiments, the composition is formulated as a wound dressingor bandage can that can provide sustained contact or release of an Erucasativa extract with a wound and inhibit the growth of Gram-positivebacteria and other microorganisms while also preventing contamination ordesiccation of a wound.

Formulations. A composition containing the Eruca sativa aqueous extractmay be provided in various forms. Formulations may include other activeingredients and/or non-toxic, inert pharmaceutically suitable excipientssuch as solid, semisolid or liquid diluents, fillers, or adjuvants.

Formulations may be in solid, semisolid or viscous, liquid, aerosol orvaporous forms. They may have acidic or basic pHs and may contain waterand/or organic ingredients such as alcohols and other conventionalorganic excipients, such as those used in pharmaceutical or cosmeticproducts. A formulation may be in the form of a solution, tincture, wash(e.g., surgical or dental washes), foam, spray, serum, gel, suppository,suspension, emulsion, cream, lotion, paste, ointment, granule, powder,freeze-dried or desiccated form, troche, capsule, tablet, or pill. Anantiseptic or cleaning formulation may contain ingredientsconventionally included in such products such as water, organiccompounds, chelators, surfactants, oxidants and disinfectants. Theparticular formulations described in detail below may contain chelatorsor antioxidants as well as other conventional excipients or carriers.

Chelators. Compositions according to the invention may incorporate oneor more chelators that can sequester elements like calcium or ironnecessary for bacterial growth. Examples of chelators of iron andcalcium include, but are not limited to, diethylene triamine pentaaceticacid (DTPA), ethylene diamine tetraacetic acid (EDTA), nitrilotriaceticacid (NTA), 1,3-propylene diamine tetraacetic acid (PDTA), Ethylenediamine disuccinic acid (EDDS), and ethylene glycol tetraacetic acid(EGTA). Any suitable chelating agent known in the art, which isbiologically safe and able to chelate iron, calcium or other metals, issuitable for the invention. Suitable biocompatible chelating agentsuseful in conjunction with the present invention include, withoutlimitation, monomeric polyacids such as EDTA, cyclohexanediaminetetraacetic acid (CDTA), hydroxyethylethylenediamine triacetic acid(HEDTA), diethylenetriamine pentaacetic acid (DTPA), dimercaptopropanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA), aminotrimethylenephosphonic acid (ATPA), citric acid, pharmaceutically acceptable saltsthereof, and combinations of any of the foregoing. Other exemplarychelating agents include: phosphates, e.g., pyrophosphates,tripolyphosphates, and hexametaphosphates. Such chelators may beincorporated in amounts sufficient to bind to the divalent cations ormetals, such as iron, in a composition or in a wound to which thecomposition is applied. For example, the concentration may be selectedto bind to at least 25, 75 or 100 mole % of the calcium, magnesium, oriron in a composition or wound.

Antioxidants. Antioxidants suitable for use in pharmaceutical, cosmetic,and food products are known. BHT (butylated hydroxytoluene) and BHA(butylated hydroxyanisole) are two common oil soluble antioxidants.Tocopherols (Vitamin E derivatives, e.g., alpha-tocopherol) and ascorbylpalmitate may also be used. Ascorbates, such as vitamin C, and propylgallate are examples of water soluble antioxidants. Alpha lipoic acid,acetyl carnitine, Coenzyme Q10 (ubiquinol), selenium, retinoic acid, Bvitamins, flavonoids, and various algae and plant extracts may also beused as antioxidants. Such antioxidants can be incorporated in amountssufficient to quench free radicals in a composition or in a wound towhich the composition is applied. For example, the concentration may beselected to bind to at least 25, 75 or 100 mole % of the free radicalsin a composition or wound.

Other Ingredients and Excipients. In addition to the activeingredient(s), the formulations described herein, depending on theirparticular physical and chemical formulation may contain the customaryexcipients, for example animal and vegetable fats, waxes, paraffins,starch, tragant or cellulose derivatives, polyethylene glycols,silicones, bentonites, silicic acid, talcum, and zinc oxide, or mixturesof these substances.

Aqueous suspensions may contain customary excipients such as liquiddiluents, for example water, ethyl alcohol, propylene glycol, suspendingagents, for example ethoxylated isostearyl alcohols, polyoxyethylenesorbit and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragant, or mixtures of thesesubstances.

The kinds of formulations mentioned herein may also contain colorants,preservatives and odor- and flavor-enhancing additives, for examplepeppermint oil and eucalyptus oil, and, for ingestible productssweeteners and flavorings. Solutions and emulsions according to theinvention may contain the customary excipients such as solvents,solubilizers and emulsifiers, for example water, ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular cottonseed oil, peanut oil, corn oil,olive oil, castor oil and sesame oil, glycerin, glycerol formal,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, or mixtures of these substances.

Some specific kinds of formulations of compositions containing anaqueous extract of Eruca sativa are described below.

Topical formulations. Topical formulations include antimicrobialpharmaceuticals, deodorants, soaps, body washes, shampoos, lubricants(e.g., for catheters or other devices, anatomical sites, or surfaces atrisk of microbial contamination), hand or skin sanitizers anddisinfectants and personal care products (e.g., a product that is usedfor personal hygiene). A topical formulation can be provided in avariety of formulations including but not limited to solutions,tinctures, gels, serums, creams, colloids, emulsions, lotions, solidsticks, aerosols or dry powders as described in U.S. Pat. Nos.4,844,902; 6,818,226; 6,469,015; 7,147,854; 7,192,607; 7,205,003; and7,252,831, each of which is hereby incorporated by reference in itsentirety.

Aqueous solutions. The Eruca sativa extract of the invention may bedissolved in water, another solvent miscible with water, or a mixturethereof. It may contain other solutes or liquid components such assalts, chelators (e.g., EDTA, EGTA), antioxidants, or preservatives. Insome embodiments it may further comprise an acid, a base, or buffer foradjusting or stabilizing the pH of an aqueous composition so as tomaintain or maximize antibacterial activity of the Eruca sativa extractor for suitability for treatment of a particular type of wound. Forexample, the acid or base is useful for adjusting the pH of the presentaqueous compositions to a pH of about 1 to about 14 (e.g., from about 1to about 2, from about 2 to about 2, from about 3 to about 4, from about4 to about 5, from about 5 to about 6, from about 6 to about 7, fromabout 7 to about 8, from about 8 to about 9, from about 9 to about 10,from about 10 to about 11, from about 11 to about 12, from about 12 toabout 13, from about 13 to about 14, or any other value or range ofvalues therein). In certain embodiments, the pH of the present aqueouscomposition ranges from about 3.5 to about 13; in other embodiments,from about 6.5 to about 8.5. In some embodiments, the pH is about 13; inother embodiments, the pH is about 7.5 to about 8.4. In certainembodiments, the pH of the present aqueous composition ranges from about5 to about 13; from about 6 to about 13; from about 7 to about 13; fromabout 8 to about 13; from about 9 to about 13; from about 10 to about13; from about 11 to about 13; from about 12 to about 13. Such pHadjustment can improve the dispersibility of ingredients present in anaqueous composition.

Acids useful in the present aqueous compositions include inorganic acidssuch as carbonic acid, sulfuric acid, or hydrochloric acid. Organicacids can alternatively be employed. Suitable organic acids include C₁to C₂₀ organic acids such as formic acid, citric acid, malic acid,adipic acid, tannic acid, lactic acid, ascorbic acid, acetic acid,fumaric acid, and mixtures thereof. In one embodiment, the acid iscitric acid. In some embodiments, the aqueous compositions do notcomprise an acid. These ranges include all intermediate values as wellas endpoints.

Bases useful in the present aqueous compositions may be organic orinorganic bases. Suitable inorganic bases include alkali metal oralkaline earth metal compounds such as sodium hydroxide, lithiumhydroxide, potassium hydroxide, sodium carbonate, potassium carbonate,sodium bicarbonate, potassium bicarbonate, magnesium carbonate andcalcium carbonate. Other suitable bases include ammonium hydroxide,substituted amine bases and ammonia. In some embodiments, the aqueouscompositions do not comprise a base. These ranges include allintermediate values as well as endpoints.

In other embodiments, the aqueous compositions can comprise one or moresalts. Salts useful in the present aqueous compositions include organicor inorganic salts. Suitable salts include alkali or alkaline earthmetal salts such as sodium chloride, sodium nitrate, potassium chloride,calcium chloride, magnesium chloride, ammonium chloride, sodium bromide,potassium bromide, calcium bromide, magnesium bromide, ammonium bromide,sodium iodide, potassium iodide, calcium iodide, magnesium iodide,ammonium iodide, sodium sulfate, potassium sulfate, calcium sulfate,magnesium sulfate, ammonium sulfate. The salt can present in the aqueouscompositions in an amount from 0 wt % to about 30 wt % 0 to about 0.5 wt%, about 0.5 wt % to about 1 wt %, about 1 wt % to about 2 wt %, about 2wt % to about 3 wt %, about 3 wt % to about 4 wt %, about 4 wt % toabout 5 wt %, about 5 wt % to about 6 wt %, about 6 wt % to about 7 wt%, about 7 wt % to about 8 wt %, about 8 wt % to about 9 wt %, about 9wt % to about 10 wt %, about 10 wt % to about 11 wt %, about 11 wt % toabout 12 wt %, about 12 wt % to about 13 wt %, about 13 wt % to about 14wt %, about 14 wt % to about wt %, about 15 wt % to about 16 wt %, about16 wt % to about 17 wt %, about 17 wt % to about 18 wt %, about 18 wt %to about 19 wt %, about 19 wt % to about 20 wt %, about 20 wt % to about21 wt %, about 21 wt % to about 22 wt %, about 22 wt % to about 23 wt %,about 23 wt % to about 24 wt %, about 24 wt % to about 25 wt %, about 25wt % to about 26 wt %, about 26 wt % to about 27 wt %, about 27 wt % toabout 28 wt %, about 28 wt % to about 29 wt %, about 29 wt % to about 30wt %, or any other value or range of values therein) of the aqueouscomposition. In some embodiments, the salt is present from about 0.01 wt% to about wt % of the aqueous compositions. In some embodiments, theaqueous compositions do not contain a salt.

In other embodiments the aqueous compositions contain water or solventsmiscible with water or a mixture of both. An aqueous composition maycontain an amount of Eruca sativa extract that exhibits antimicrobial ortherapeutic (e.g., wound healing, etc.) activity in combination with anamount of solvent sufficient to keep the extract in solution or todissolve a dry extract. In some embodiments, the amount of water oraqueous solvent in the present aqueous compositions can range from about10 to about 90 wt % (e.g., about 10 wt % to about wt %, about 15 wt % toabout 20 wt %, about 20 wt % to about 25 wt %, about 25 wt % to about 30wt %, about 30 wt % to about 35 wt %, about 35 wt % to about 40 wt %,about 40 wt % to about 45 wt %, about 45 wt % to about 50 wt %, about 50wt % to about 55 wt %, about wt % to about 60 wt %, about 60 wt % toabout 65 wt %, about 65 wt % to about 70 wt %, about 70 wt % to about 75wt %, about 75 wt % to about 80 wt %, about 80 wt % to about 85 wt %,about 85 wt % to about 90 wt %, or any other value or range of valuestherein). In certain embodiments, the aqueous compositions comprise fromabout 80 wt % to about 90 wt % water or about 90 wt. % to about <100 wt.% water.

The present aqueous compositions can further comprise an organicsolvent, in the absence or presence of water in an amount sufficient tokeep the extract in solution or to dissolve a dry extract. Suitableorganic solvents include, but are not limited to, C₁ to C₄ alcohols suchas methanol, ethanol, n-propanol and i-propanol, n-butanol, sec-butanol,isobutanol and tert-butanol. Alternatively, glycols such as ethyleneglycol, propylene glycol and polyethylene glycol, and ketone-containingsolvents such as acetone can be employed. In certain embodiments, theaqueous organic solvent is ethanol or i-propanol. In one embodiment, theaqueous compositions comprise water and an alcohol; in anotherembodiment, water and ethanol or i-propanol. The amount of organicsolvent, if present, can be selected based on factors such as itsmiscibility in water, if present, and the amount of Eruca sativaextract. In some embodiments, the organic solvent can be present in theaqueous compositions in an amount ranging from 0 wt % to about 10 wt %(e.g., 0 wt % to about 1 wt %, about 1 wt % to about 2 wt %, about 2 wt% to about 3 wt %, about 3 wt % to about 4 wt %, about 4 wt % to about 5wt %, about 5 wt % to about 6 wt %, about 6 wt % to about 7 wt %, about7 wt % to about 8 wt %, about 8 wt % to about 9 wt %, about 9 wt % toabout 10 wt %, or any other value or range of values therein) of theaqueous composition. In some embodiments, the aqueous compositions donot comprise an organic solvent.

The aqueous compositions of the invention can further contain one ormore other additives. Suitable additives include, but are not limitedto, detergents, as surface tension modifiers, flocculants, dispersants,rheology modifiers, emulsifiers, surfactants, chelators, and solvents.Illustrative additives are polysorbates, oils (e.g., canola oil,vegetable oils, etc.). In some embodiments, the additive(s) can bepresent in the aqueous compositions in amounts ranging from 0 to about30 wt % (e.g., 0 to about 0.5 wt %, about 0.5 wt % to about 1 wt %,about 1 wt % to about 2 wt %, about 2 wt % to about 3 wt %, about 3 wt %to about 4 wt %, about 4 wt % to about 5 wt %, about 5 wt % to about 6wt %, about 6 wt % to about 7 wt %, about 7 wt % to about 8 wt %, about8 wt % to about 9 wt %, about 9 wt % to about 10 wt %, about 10 wt % toabout 11 wt %, about 11 wt % to about 12 wt %, about 12 wt % to about 13wt %, about 13 wt % to about 14 wt %, about 14 wt % to about 15 wt %,about 15 wt % to about 16 wt %, about 16 wt % to about 17 wt %, about 17wt % to about 18 wt %, about 18 wt % to about 19 wt %, about 19 wt % toabout 20 wt %, about 20 wt % to about 21 wt %, about 21 wt % to about 22wt %, about 22 wt % to about 23 wt %, about 23 wt % to about 24 wt %,about 24 wt % to about 25 wt %, about 25 wt % to about 26 wt %, about 26wt % to about 27 wt %, about 27 wt % to about 28 wt %, about 28 wt % toabout 29 wt %, about 29 wt % to about 30 wt %, or any other value orrange of values therein) of the aqueous composition.

In some embodiments, the present aqueous compositions will contain asurfactant. Surfactants are compounds that lower the surface tension ofa liquid, the interfacial tension between two liquids, or that between aliquid and a solid. Surfactants may act as detergents, wetting agents,emulsifiers, foaming agents, and dispersants. Surfactants that can bepresent in the compositions of the invention include anionicsurfactants, amphoteric surfactants, cationic surfactants, zwitterionicsurfactants, non-ionic surfactants, and combinations thereof.Surfactants suitable for use in the present invention can includepolysorbates such as polysorbate 20, polysorbate 40, polysorbate 60 orpolysorbate 80. An amount of surfactant that is sufficient to emulsifyother ingredients in an aqueous composition or that is sufficient tolower surface tension of a pharmaceutical composition so as to maximizecontact between a wound and the Eruca sativa extract or maximizeantimicrobial activity may be selected.

Serums. A serum refers to a light, quickly absorbed composition thatexposes and permits rapid uptake of an active ingredient by skin. It canbe used as an alternative to heavier creams or lotions that containocclusive, or airtight, moisturizing ingredients such as petrolatum ormineral oil that keep water from evaporating. Serums usually containfewer lubricating and thickening agents, like nut or seed oils, thancreams or lotions. Most serums are water-based or based on hydrophiliccomponents, eliminating oils altogether. A serum may be formulated tocontain a higher concentration of an active ingredient, such as Erucasativa extract, than a cream or lotion.

Gels. Gels provided herein include semi-solid suspensions that containan Eruca sativa extract. The gels can be single- or two-phase systems.The gels can be oil or liquid based. Single-phase gels can contain smallorganic macromolecules distributed substantially uniformly throughout aliquid, such that the there is no boundary between the macromoleculesand liquid. The liquid can be aqueous, but also contain an alcohol, and,optionally, an oil. Single-phase gels can be made from syntheticmacromolecules or from natural gums. Two-phase gels can include anetwork of small, discrete particles. In one embodiment, two-phase gelsare thixotropic. In one embodiment, the organic macromolecules includecrosslinked acrylic acid polymers such as the “carbomer” family ofpolymers (i.e., carboxypolyalkylenes). The organic macromolecules canalso be hydrophilic polymers such as polyethylene oxides,polyoxyethylene-polyoxypropylene copolymers and polyvinyl alcohol;cellulosic polymers such as hydroxypropyl cellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate, and methyl cellulose; gums such as tragacanth and xanthangum; sodium alginate; and gelatin. In another embodiment, the organicmacromolecules having a stabilizing action include long-chain linearhigh molecular weight polysaccharides with a molecular weight of morethan one million. In this embodiment, 0.1 to 1.5 wt % of suchstabilizers are included. In another embodiment, a uniform gel can beprepared by adding dispersing agents such as alcohol or glycerin. Inanother embodiment, the organic macromolecules can be dispersed bytrituration, mechanical mixing or stirring, or combinations thereof. Inanother embodiment, the liquid can be either water or all water-misciblesolvents. Examples of applicable solvents include alkanols, such asethanol and isopropyl alcohol, benzyl alcohol, propylene glycol andsimilar solvents.

Creams/Emulsions. Creams provided herein include liquids or semi-solidemulsions with a viscous consistency. Creams can be either oil-in-wateror water-in-oil based formulations. Cream bases can be water soluble.Cream bases can contain the following components: (1) an oil phase, (2)an aqueous phase, and (3) an emulsifier. The oil phase can comprisepetroleum jelly and a fatty alcohol, such as cetyl or stearyl alcohol.The aqueous phase can contain a humectant. The emulsifier can be anonionic, anionic, cationic or amphoteric surfactant. In one embodiment,the oil phase includes, but is not limited to, cetyl alcohol, stearylalcohol, stearic acid, liquid paraffin, and dimethicone. In anotherembodiment, the water phase ingredient includes, but is not limited to,glycerol and ethyl paraben as well as Eruca sativa aqueous extract. Inanother embodiment, the emulsifying agent includes, but is not limitedto, fatty alcohol polyoxyethylene ether (Peregal A-20), polyoxylstearate(SG-6), or combinations thereof.

Lotions. Lotions provided herein include liquids or semi-liquidformulations that are generally lower in viscosity than a cream or gel.The lotions can be an oil-in-water formulation stabilized by asurface-active agent and are usually suitable for application tounbroken skin. In one embodiment, the lotions contain suspending agentsto produce better dispersions and compounds useful for localizing andholding the active agent such as components of an Eruca sativa aqueousextract in contact with the skin, including methylcellulose, sodiumcarboxymethyl-cellulose, and similar compounds.

Ointments. Ointments provided herein include semi-solid preparationsthat have petroleum jelly or their derivatives as a base. Petroleumjelly is a semi-solid mixture of hydrocarbons. As described inRemington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.:Mack Publishing Co., 1995), at pages 1399-1404, ointment bases can begrouped in four classes: oleaginous bases; emulsifiable bases; emulsionbases; and water-soluble bases. Oleaginous ointment bases includevegetable oils, fats obtained from animals, and semisolid hydrocarbonsobtained from petroleum. Emulsifiable ointment bases, also known asabsorbent ointment bases, contain little or no water and include, forexample, hydroxystearin sulfate, anhydrous lanolin, and hydrophilicpetroleum jelly. Emulsion ointment bases are either water-in-oil oroil-in-water emulsions, and include, for example, cetyl alcohol,glyceryl monostearate, lanolin, and stearic acid. An ointment maycontain solid or encapsulated particles of a dry Eruca sativa aqueousextract or may contain an emulsified or suspended Eruca sativa aqueousextract.

Pastes. Pastes included herein contain, in addition to an ointment orcream base, high amounts of pulverulent constituents, such as zincoxide, talc, starch or titanium dioxide. In one embodiment, the paste isselected from the group comprising fatty pastes or single-phase aqueousgels. The fatty paste includes petroleum jelly, hydrophilic petroleumjelly, or other similar compounds. The single-phase aqueous gel canincorporate carboxymethylcellulose or similar compounds. A paste maycontain solid or encapsulated particles of a dry Eruca sativa aqueousextract or may contain an emulsified or suspended Eruca sativa aqueousextract.

Aerosols. Aerosol as provided herein includes products packaged underpressure and contain ingredients that are released upon activation of anappropriate valve system. Aerosols include all self-containedpressurized products, such as fine mists of spray or foam, that areemitted from a pressurized container containing a propellant, foams, orsemisolid liquids. They may also be emitted by an unpressurized atomizerthat is pressurized by a hand-operated pump rather than by storedpropellant. In one embodiment, the aerosol comprises a container, apropellant, a concentrate containing an active ingredient, a valve(which may be a metered valve), and an actuator. The nature of thesecomponents determines characteristics such as delivery rate, foamdensity, and fluid viscosity. In another embodiment, the aerosol is atwo-phase formulation comprising a gas and liquid. In anotherembodiment, the aerosol is a three-phase formulation comprising a gas,liquid, and suspension or emulsion of active ingredients. In thisformulation, suitable excipients, such as wetting agents and/or solidcarriers such as talc or colloidal silicas are included. In anotherembodiment, the propellant is liquefied or vaporized. In anotherembodiment, a solvent can be the propellant or a mixture of thepropellant and co-solvents such as alcohol and polyethylene glycols. Inanother embodiment, the propellant is selected from the group comprisinga spray, foam, or quick-breaking foam. In another embodiment, sprayformulations are aqueous solutions in a container having a spray means,such as an atomizer or nebulizer. A spray may contain an aerosol ofsolid or encapsulated particles of a dry Eruca sativa aqueous extractcomposition or may contain an Eruca sativa aqueous extract in anatomized or aerosol liquid form.

Foams. In some embodiments, an Eruca sativa aqueous extract is deliveredto the body while in a foam state, such as stable foam, for example,that is produced with or without a propellant. For example, the extractmay be contained in a shaving foam and used for preventing bacterialinfection of nicks, cuts or abrasions associated with shaving. In someversions, a foam is dispensed from a dispenser such as a propellant-freedispenser with pumping action to create the foam from a composition in afoamable carrier, and then applied to a wipe or other substrate, orapplied to the hand of the user or otherwise delivered to the skin.Propellant-driving foam generators may also be used to deliver thecomposition in the form of a foam. Active ingredients in a foam may bedispensed for subsequent placement on a dry wipe, a pre-moistened wipe,or other soft, flexible applicator (e.g., an object about 3-fingers wideor 4 to 10 cm wide) or other object to be used for application of thefoam-based composition to the skin. The foam can be a non-propellantfoam. A foam with a suitable stiffness of yield stress can be applied tothe skin in any manner for sustained adherence and contact with thebody. Examples of foam-based systems are described in U.S. Pat. No.6,818,204, “Stable Foam for Use in Disposable Wipe,” issued to Lapiduson Nov. 16, 2004, herein incorporated by reference. The Lapidus patentinvolves the use of compatible surfactants, e.g., nonionic, anionic,amphoteric, for use in human hygienic products. The surfactant should becapable of forming a foam when mixed with air in a finger actuated,mechanical pump foamer. Such surfactants are said to include, withoutlimitation, those which do not irritate mucous membranes such aspolyethylene cetyl ether (Brij 58)™, a nonionic surfactant; sodiumlauroyl sarcosinate (Hamposyl L-sodium lauryl sulfoacetate (LathanolLAL)™ and sodium laureth sulfate (Sipon ESY)™, anionic surfactants;lauramidopropyl betaine (Monateric LMAB™), an amphoteric surfactant, aswell as polysorbate 20, TEA-cocoyl glutamate, disodiumcocoamphodiacetate and combinations thereof. Typically, the surfactantis said to present in an amount from about 2% to about 35% by weight, orfrom about 5% to about 15% by weight.

At least one foam stabilizing agent may be present in some foamableembodiments. Suitable foam stabilizing agents may include, withoutlimitation, natural or synthetic gums such as xanthan gum, polyalkyleneglycols such as polyethylene glycol, alkylene polyols such as glycerineand propylene glycol and combinations thereof. Typically, the foamstabilizers may be present in an amount from about 0.10% to about 5%, orfrom about 2% to about 4%. In the Lapidus patent (U.S. Pat. No.6,818,204), alkylene polyols are said to be typically employed inamounts from about 0.1% to about 10%, gums are employed in amountsranging from about to about 1%, and/or polyalkylene glycols are presentin amounts ranging from about to about 2%.

A foam may be produced using the F2 Finger Pump Foamer™ manufactured byAirSpray International Inc. of Pompano Beach, Fla. Such a spring-loadedvalve system operates without the use of gas propellants or the like.Upon actuation, precise amounts of air and liquid are mixed, and a foamcapable of maintaining its structure for a substantial length of time isdispensed. In addition, the dispenser can deliver a variable amount offoam, thereby reducing waste of the wipe agent contained therein.Details of exemplary propellantless defoamers are described in U.S. Pat.No. 5,443,569, issued on Aug. 22, 1995, and U.S. Pat. No. 5,813,576,issued Sep. 29, 1998, herein incorporated by reference.

Other ingredients in topical formulations. The topical formulationsprovided herein can include additional ingredients to affect thephysical or functional characteristics of the formulations. Stabilizers,preservatives, humectants, regreasing agents, solvents or auxiliariescan be included to improve efficacy and dermal penetration. Dermalpenetration-enhancing compounds provided have low toxicity to the skinand can promote percutaneous and oral mucosal absorption. In oneembodiment, dermal penetration-enhancing compounds include propyleneglycol, polyethylene glycol, dimethylsulphoxide, decylmethylsulphoxide,azones, N-methylpyrrolidone, diethyltoluamide, ethanol, isopropylmyristate, isopropyl palmitate, oleic acid and its esters, medium-chaintriglycerides, dimethyl isosorbitol, 2-octyldodecanol, branched fattyacids, benzyl alcohol, urea, salicylates and surfactants. Viscosityenhancers or thickeners can be included. Such enhancers can prevent theformulation from spreading beyond the site of application. In oneembodiment, Balsam Fir is a pharmaceutically acceptable viscosityenhancer. Another benefit of increasing the viscosity of the formulationis provided below in the section discussing thixotropic agents.Thickeners include suitable polymers such as carbomer, hydroxypropylmethylcellulose, hydroxyethylcellulose, PVM/MA decadiene cross-polymerand acrylates. Two or more thickeners can be added.

Spreading oils or emollients can be included. One benefit for includingsuch oils is for better distribution on surfaces, in particular on theskin. Spreading oils are understood as those oily liquids which aredistributed particularly easily on the skin. They are known as such incosmetics. The following compounds are suitable spreading agents:silicone oil, fatty acid esters, such as ethyl stearate, di-n-butyladipate, hexyl laurate and dipropylene glycol pelargonate, esters of abranched fatty acid of medium chain length with saturated C₁₆-C₁₈ fattyalcohols, isopropyl myristate, isopropyl palmitate, caprylic/capric acidesters of saturated fatty alcohols of C₁₂-C18 chain length, isopropylstearate, oleyl oleate, decyl oleate, ethyl oleate, ethyl lactate, waxyfatty acid esters, such as synthetic duck uropygial gland fat, dibutylphthalate, diisopropyl adipate, ester mixtures related to the latter andthe like. Other elements that can be included are emollients, suchdiisopropyl adipate/isohexadecane dimethicone, occlusive agents, such asexample cyclomethicone, trimethylsiloxysilicate, glycereth-26 orpolyquaternium-7, emulsifiers, such as cetyl alcohol, stearyl, stearicacid, glyceryl stearate, propylene glycol isostearoyl-sodiumisostearoyl, a lactylate, polyoxyethylene (100) stearate, skinconditioners, moisturizers, humectants, such as propylene glycol orglycerin, preservatives, such as phenoxyethanol and parabens, pHadjusting agents, surfactants, chelators, such as disodium EDTA orsodium citrate, tackifying agents, fragrances and other compounds.

Other compounds that can be included in the topical formulation includeother antimicrobial, antibacterial, anti-inflammatory ingredients, orother functional ingredients such as protectants from UV. These include,but not limited to, antibiotics that target Gram-negative orGram-positive bacterial, antifungal compounds, NSAIDS, glucocorticoids(e.g., hydrocortisone and derivatives having the same core ringstructure), benzyl alcohol, other botanical extracts or oils, such asthose described herein.

The compositions of this invention may be used in conjunction with otheractive ingredients, such as phytochemicals or non-Eruca sativa extracts,bacteriostatic agents, bactericidal agents, antibiotics, antiseptics, oranti-inflammatory agents.

Encapsulation. The Eruca sativa extracts described herein can beencapsulated in a carrier such as in liposomes, micelles, ormicrospheres. Suitable carriers are described in U.S. Pat. No.7,205,003, hereby incorporated by reference.

Micelles. Micelles provided herein can comprise surfactant moleculesarranged such that their polar head groups form an outer sphericalshell, while their hydrophobic, hydrocarbon chains are oriented towardsthe center of the sphere, forming a core. The precursor and agent areencapsulated within the core of the micelle. Surfactants suitable forforming micelles include, but are not limited to, potassium laurate,sodium octane sulfonate, sodium decane sulfonate, sodium dodecanesulfonate, sodium lauryl sulfate, docusate sodium,decyltrimethylammonium bromide, dodecyltrimethylammonium bromide,tetradecyltrimethylammonium bromide, tetradecyltrimethyl-ammoniumchloride, dodecylammonium chloride, polyoxyl-8 dodecyl ether,polyoxyl-12 dodecyl ether, nonoxynol 10, and nonoxynol 30.

Liposomes. Liposomes provided herein are microscopic vesicles having alipid wall comprising a lipid bilayer. Liposomal preparations hereininclude cationic (positively charged), anionic (negatively charged), andneutral preparations. Cationic liposomes includeN[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA). Anionic andneutral liposomes can be easily prepared using materials such asphosphatidyl choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), and dioleoylphoshatidyl ethanolamine (DOPE). These materials canalso be mixed with DOTMA in appropriate ratios.

Microspheres. Microspheres provided herein can comprise micro- ornano-scale carriers that are made of polymers, both synthetic andnatural. Additional nomenclature describing microspheres include, butare not limited to, spheres, beads, particles, carriers, microbeads,microparticles, microcarriers, nanospheres, nanobeads, nanoparticles,and nanocarriers.

Polymeric materials suitable for the microspheres provided hereininclude those that are described in U.S. Pat. No. 6,423,345, herebyincorporated by reference in its entirety for all purposes, includingpoly(hydroxy acids) such as poly(lactic acid), poly(glycolic acid), andpoly(lactic acid-co-glycolic acid), poly(lactide), poly(glycolide),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, polyamides,polycarbonates, polyalkylenes such as polyethylene and polypropylene,polyalkylene glycols such as poly(ethylene glycol), polyalkylene oxidessuch as poly(ethylene oxide), polyalkylene terepthalates such aspoly(ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers,polyvinyl esters, polyvinyl halides such as poly(vinyl chloride),polyvinylpyrrolidone, polysiloxanes, poly(vinyl alcohols), poly(vinylacetate), polystyrene, polyurethanes and co-polymers thereof,derivativized celluloses such as alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt (jointly referred to herein as “syntheticcelluloses”), polymers of acrylic acid, methacrylic acid or copolymersor derivatives thereof including esters, poly(methyl methacrylate),poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate),poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), andpoly(octadecyl acrylate) (jointly referred to herein as “polyacrylicacids”), poly(butyric acid), poly(valeric acid), andpoly(lactide-co-caprolactone), copolymers and blends thereof. As usedherein, “derivatives” include polymers having substitutions, additionsof chemical groups, for example, alkyl, alkylene, hydroxylations,oxidations, and other modifications routinely made by those skilled inthe art. Natural polymers including agarose and alginate are alsosuitable for the microspheres. Any of the above carriers can includeproteins, lectins, and other biological materials. The precursors andactivating agents can be encapsulated into the carriers using knowntechniques in the art, including microspheres described in U.S. Pat. No.6,423,345, incorporated by reference, including solvent evaporation, hotmelt microencapsulation, solvent removal, and spray drying ofmicrospheres. In one embodiment, the microsphere comprises a blockcopolymer. In another embodiment, the microsphere comprises a hydrogel.

Suppositories. In addition to the active Eruca sativa extract, asuppository may contain the customary water-soluble or water-insolubleexcipients, for example polyethylene glycols, fats, for example cocoafat and higher esters (e.g., C₁₄-alcohol with C₁₆-fatty acid) ormixtures of these substances.

Tablets, Capsules, Pills. In some embodiments, the Eruca sativa aqueousextract will be formulated as a tablet, capsule or pill that contains anEruca sativa aqueous extract. These may contain the customaryexcipients, such as fillers and extenders, for example starches,lactose, sucrose, glucose, mannitol, and silicic acid; binders, forexample carboxymethyl cellulose, alginates, gelatin,polyvinylpyrrolidone; humectants, for example glycerin; disintegratingagents, for example agar-agar, calcium carbonate and sodium carbonate;dissolution retardants, for example paraffin; resorption acceleratingagents, for example quaternary ammonium compounds; wetting agents, forexample cetyl alcohol, glycerol monostearate; adsorption agents, forexample kaolin and bentonite; and lubricants, for example talcum,calcium stearate and magnesium stearate, and solid polyethylene glycolsor mixtures of the substances mentioned above. In some embodiments, theactive ingredient(s) can be in a microencapsulated form in the tablet orcapsule, which can optionally be formulated to release the active Erucasativa component at a particular location within the GI tract, e.g, totransit the stomach and release the active component in the small orlarge intestine.

Powder. A composition according to the invention may be formulated inthe form of an antimicrobial powder which contains a dry or encapsulatedEruca sativa extract and the customary excipients, for example lactose,talcum, silicic acid, aluminum hydroxide, calcium silicate, andpolyamide powder, or mixtures of these substances. Such powders may beformulated for topical application or for inhalation.

Deodorant/Personal Care. The compositions of the invention can beformulated as deodorants or personal care products that prevent theformation of body odors such as those produced by the growth ofGram-positive bacteria in or on the skin. Such deodorant will contain anantimicrobial amount of the Eruca sativa extract and suitable excipientsor carriers to facilitate its application to the body. Eruca sativaextract may be incorporated into conventional body washes, lotions,lubricants, personal care composition, antiperspirants, or deodorants.Such products are well known in the art and commercially available andare also described by Broad, U.S. Pat. No. 4,252,789, which isincorporated by reference, especially for their descriptions ofconventional deodorant ingredients, formulations, and modes of use.These products can be applied to the axilla, inguinal region, feet orother odor-producing body part to prevent growth of odor-causingmicroorganisms. In other embodiments, the extract of the invention canbe incorporated into a deodorizer, cleaner, or disinfectant such as aliquid sanitizer or disinfectant, a spray or wipe for cleaning surfacesexposed to bacterial contaminants.

Cleaning Agent. A composition containing an Eruca sativa aqueous extractmay be formulated for use as a cleaning or disinfecting agent, such as ahard surface cleaning product. It may further contain cleaning agents,such as chelators or surfactants, which do not interfere with theantimicrobial activity of the Eruca sativa extract. The formulation ofsuch a cleaning or disinfecting solution and inclusion of generalcleaning agents can easily be done by a skilled artisan and thestability and effectiveness of the solution can be easily tested by theskilled artisan. The term “hard surface cleaning composition” refers toa composition that is used to clean and/or sanitize a hard or solidsurface. In one embodiment, the invention provides a composition thatprevents bacterial growth on hard surfaces including, but not limitedto, surgical instruments, storage tanks, pipelines, trays, containers,walls, floors, countertops, locker room floors, benches, lockers,showers, bathrooms, toilets, water filtration units, and the like.

Foods, Beverages & Feeds. In other embodiments, the formulation is inthe form of an ingestible food or beverage or in an additive orprotectant in aquaculture. It may be a human food or animal feed thatcontains an Eruca sativa aqueous extract. Such extracts may beincorporated into animal feeds for mammals (cattle, sheep, goats, etc.),birds (e.g., chickens, turkeys, quail, ducks, geese, hawks, falcons,etc.), fish (e.g., tilapia, carp, catfish, salmon, trout, aqua culturedfish, etc.), and crustaceans (e.g., shrimp, lobsters, etc.), mollusks(e.g., abalone, oysters, clams, mussels, etc.). In some embodiments, theEruca sativa extract of the invention will be incorporated into a liquidmedium in which an animal is grown, e.g., into a medium for aquaculture.In other embodiments, the extract may be encapsulated in a form thatpermits uptake by an animal, for example, in an encapsulated particulateform that can be ingested by fish.

Plants. In some embodiments, the Eruca sativa extract of the inventioncan be applied to control the growth of Gram positive bacteria in or onplants, such those causing leaf spots, rots, scabs, and wilting. It maybe sprayed or otherwise applied to the roots, foliage, flowers or seedsof a plant. It may be added to culture medium used for hydroponiccultivation of plants.

In many embodiments, the Eruca sativa aqueous extract will exertantimicrobial activity one anti-Gram positive bacteria. Antimicrobialactivity may be determined or quantified by assays known in the art.Representative assays are described below.

Antimicrobial Assays. The term “antimicrobial” when used in the contextof an antimicrobial agent or antimicrobial composition, refers to anagent or composition that can kill or otherwise inhibit the growth orproliferation of microbes including, for example, bacteria, viruses andfungi. Similarly, the term “antimicrobial activity” as used hereinrefers to activity that can kill or otherwise inhibit the growth orproliferation of microbes including bacteria, yeasts, viruses and fungi.

When used as an antimicrobial agent, those skilled in the art understandthat it is beneficial to select extraction, treatment, purification, andstorage conditions of the Eruca sativa aqueous extract to maximize theanti-microbial or therapeutic activity of a composition containing theextract, for example, by selecting an appropriate concentration of theextract. These selections may be made based on the results of theantimicrobial assays described below.

The antimicrobial or antibacterial activity of the Eruca sativa extractof the invention may be determined using standard assays and proceduresknown in the art. A skilled artisan will know how to conduct an assayincluding the use of appropriate controls, negative and positive, toensure the results of the assay are meaningful. Common antimicrobialassays include the broth dilution and agar diffusion assays.

In the broth dilution method, the minimum concentration required toinhibit bacterial growth (minimum inhibitory concentration, MIC) isdetermined using a series of tubes containing serial dilutions of theextract in broth inoculated with a test microorganism.

In the disc diffusion method, paper or porous discs impregnated with atest extract are placed on a semi-solid (agar based) medium which hasbeen uniformly inoculated with a microorganism. After incubation, zonesof inhibition of bacterial growth around the discs are measured.Inhibition may be quantified by reference to that caused by controldiscs containing known concentrations of antimicrobial compounds such asantibiotics. Disc diffusion assays are incorporated by reference toMohanty A, et al, Physiochemical and Antimicrobial Study of polyherbalPharmacieglobal, 4 (04): 1-3 (2010) or to the Kirby-Bauer methoddescribed by hypertext transferprotocol://web.archive.org/web/20130731092046/and hypertext transferprotocol://aminj.myweb.uga.edu:80/KIRBY-BAUER.html (last accessed Mar.28, 2017).

Standardization of Eruca sativa extract microbial activity. Theantimicrobial activity of an aqueous Eruca sativa extract may bedetermined by its effect on a bacterial strain, such the B22Cellulomonas uda reference strain or another Cellulomonas strain such asCellulomonas sp. ATCC 21399 described as follows and incorporated byreference to hypertext transfer protocolsecure://www.atcc.org/products/all/21399.aspx (last accessed Jun. 6,2017):

-   -   Cellulomonas sp.    -   21399™    -   Description    -   Strain designation: [NCIB 11494]    -   Deposited As: Cellulomonas sp.    -   Type strain: No    -   Patent depository: This material was deposited with the ATCC        Patent Depository to fulfill U.S. or international patent        requirements. This material may not have been produced or        characterized by ATCC. As an International Depository Authority        (IDA) for patent deposits, ATCC is required to complete        viability testing only at time of initial deposit of patent        material. Patent deposits are made available on behalf of the        Depositor when the pertinent U.S. or international patent is        issued, but material may not be used to infringe the patent        claims.    -   Patent number: 3,627,095—incorporated herein by reference.    -   Technical information: ATCC Technical Services does not have        technical information on patent deposits that are not produced        or characterized by ATCC. Additional information can be found in        the corresponding patent available from the patent holder or        with the U.S. and/or international patent office.    -   Storage Conditions    -   Product format: Freeze-dried    -   Intended Use: This product is intended for laboratory research        use only. It is not intended for any animal or human therapeutic        use, any human or animal consumption, or any diagnostic use.    -   BSL 1—ATCC determines the biosafety level of a material based on        our risk assessment as guided by the current edition of        Biosafety in Microbiological and Biomedical Laboratories (BMBL),        U.S. Department of Health and Human Services. It is your        responsibility to understand the hazards associated with the        material per your organization's policies and procedures as well        as any other applicable regulations as enforced by your local or        national agencies. ATCC highly recommends that appropriate        personal protective equipment is always used when handling        vials. For cultures that require storage in liquid nitrogen, it        is important to note that some vials may leak when submersed in        liquid nitrogen and will slowly fill with liquid nitrogen. Upon        thawing, the conversion of the liquid nitrogen back to its gas        phase may result in the vial exploding or blowing off its cap        with dangerous force creating flying debris. Unless necessary,        ATCC recommends that these cultures be stored in the vapor phase        of liquid nitrogen rather than submersed in liquid nitrogen.    -   Growth Conditions    -   Medium:    -   ATCC Medium 464: Cellulomonas PTYG medium    -   Temperature: 30° C.    -   Handling Procedures    -   1. Open vial according to enclosed instructions.    -   2. Using a single tube of #464 broth (5 to 6 ml), withdraw        approximately 0.5 to 1.0 ml with a Pasteur or 1.0 ml pipette.        Rehydrate the entire pellet.    -   3. Aseptically transfer this aliquot back into the broth tube.        Mix well.    -   4. Use several drops of the suspension to inoculate a #464 agar        slant and/or plate.    -   5. Incubate the tubes and plate at 30° C. for 24 hours.    -   Notes—Colonies on #464 agar are entire, glistening, circular,        smooth, low convex, and opaque, becoming yellowish with age;

or to the other commercially available Cellulomonas strains available atthe ATCC which are incorporated by reference to the link below lastaccessed Jun. 6, 2017: hypertext transfer protocolsecure://www.atcc.org/Search Results.aspx?dsNav=Ntk:PrimarySearch %7cCellulomonas+sp.%7c3%7c,Ny: True,Ro: 0,N: 1000552&searchTerms=Cellulomonas+sp.&redir=1.

For example, the dried aqueous extracts described in Examples 1-4 can bedissolved in water or in a suitable buffer and applied at a specificconcentration to a disc for use in a disc-diffusion assay or seriallydiluted for use in a broth dilution assay. A liquid Eruca sativa extractor a composition containing an Eruca sativa aqueous extract may betested in a similar way and the dry or solid content of such a liquidextract used to standardize comparisons of liquid extracts at differentconcentrations.

“Gram-positive bacteria” are bacteria that give a positive result in theGram stain test. Gram-positive bacteria take up the crystal violet stainused in the test, and then appear to be purple-colored when seen througha microscope. This is because the thick peptidoglycan layer in thebacterial cell wall retains the stain after it is washed away from therest of the sample, in the decolorization stage of the test. In general,the following characteristics are present in gram-positive bacteria (i)a cytoplasmic lipid membrane, (ii) a thick peptidoglycan layer, (iii)teichoic acids and lipoids are present, forming lipoteichoic acids,which serve as chelating agents, and also for certain types ofadherence, (iv) peptidoglycan chains that are cross-linked to form rigidcell walls by a bacterial enzyme DD-transpeptidase, (v) a much smallervolume of periplasm than that in gram-negative bacteria.

Representative Gram-positive bacteria include bacilli and cocci. Bacilliinclude Corynebacteria, Clostridium, Listeria and Bacillus. Cocciinclude Staphylococcus and Streptococcus. Representative species includeStaphylococcus aureus (including MRSA), Staphylococcus epidermidis, andStaphylococcus saprophyticus; and Streptococcus pyogenes, Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus viridans,Enterococcus faecalis, Enterococcus faecium. In preferred embodiments,the Eruca sativa aqueous extract will exert antimicrobial activity onone or more of the Gram positive bacteria described above, such asCellulomonas strain B22. Cellulomonas is a genus of Gram-positiverod-shaped bacteria. One species is Cellulomonas uda (incorporated byreference to hypertext transferprotocol://www.uniprot.org/taxonomy/1714, last accessed Jun. 6, 2017).Other Cellulomonas species and their features are incorporated byreference to hypertext transferprotocol://www.bacterio.net/cellulomonas.html (last accessed Jun. 6,2017) and to the references cited there. One of their maindistinguishing features is their ability to degrade cellulose, usingenzymes such as endoglucanase and exoglucanase. They are members of theactinobacteria. Extracts according to the invention may be employed toinhibit growth or activity, such as enzyme activity, of Gram-positivebacteria.

Other Gram-positive bacteria which are plant pathogens includeBurkholderia, and Proteobacteria such as Xanthomonas spp. andPseudomonas spp. The extract of the invention may be used, for example,by application to plant surfaces, to control such plant pathogens.

“Mollicutes” and “Mycoplasma” describe bacteria that lack a cell wallaround their cell membrane. These include plant pathogens such asPhytoplasma and Spiroplasma. Without a cell wall, they are unaffected bymany common antibiotics such as penicillin or other beta-lactamantibiotics that target cell wall synthesis. Mycoplasma species thatinfect humans include M. amphoriforme, M. buccale, M. faucium, M.fermentans, M. genitalium, M. hominis, M. hpophilum, M. orale, Mpenetrans, M. pirum, M pneumoniae, M. primatum, M. sahvarium, and M.spermatophilum. Phylogenetically, mycoplasmas evolved from Gram-positivebacteria which lost their cell walls. They are associated with cancer(e.g, colon cancer, gastric cancer, lung cancer, prostate cancer, andrenal cancer) and host cell transformation as well as with infectiousand autoimmune diseases including genital infections and sexuallytransmitted diseases, pelvic inflammatory disease, male infertility, andpneumonia. In some embodiments, the Eruca sativa aqueous extract willexert antimicrobial activity on one or more types of mycoplasmadescribed above.

“Gram-negative bacteria” are bacteria that give a negative result in theGram stain test because they do not retain the crystal violet stain usedin the Gram staining method of bacterial differentiation. They arecharacterized by their cell envelopes, which are composed of a thinpeptidoglycan cell wall sandwiched between an inner cytoplasmic cellmembrane and a bacterial outer membrane. Gram-negative bacteria includethe following genuses and species: Acinetobacter (e.g., A. baumannii),Chlamydia (e.g., C. trachomatis), Escherichia (e.g. E. coli),Haemophilus (e.g., H. influenzae), Klebsiella (e.g., K. pneumoniae),Neisseria (e.g., N. gonorrhoeae), Pseudomonas (e.g., P. aeruginosa),Salmonella (e.g., S. enterica, S. typhi), and Yersinia (e.g., Y.pestis). In some embodiments, the Eruca sativa aqueous extract willexert no or minimal antimicrobial activity on one or more of the Gramnegative bacteria described above.

Embodiments of the invention include, but are not limited to thefollowing.

A composition comprising an aqueous extract of Eruca sativa leaves fromwhich solid components of Eruca sativa have been removed. In oneembodiment the aqueous extract is produced by grinding, pressing,pulverizing, or otherwise disrupting leaves of Eruca sativa, mixing theground, pulverized, pressed or otherwise disrupted leaves with water fora time sufficient to extract water-soluble components, and removingsolid components from the mixture of leaves and water, thereby providingthe aqueous extract. Solids may be removed by centrifugation, filtrationor by other methods known in the art. In one embodiment the mixing isperformed at a pH ranging from 6.0 to 8.0 and at a temperature rangingfrom 25° C. to 80° C.

The aqueous extract from Eruca sativa leaves may be concentrated to lessthan its original volume by removing the aqueous solvent or water. Forexample, it may be reduced to 99, 95, 90, 80, 70, 60, 50, 40, 30, 20,10, 5, 4, 3, 2, 1 of its original volume; or 5, 10, 15, 20, 25, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100 wt % of the wateror solvent may be removed. All of the solvent or water may be removedfrom the aqueous extract to produce an anhydrous or solid aqueousextract. This advantageously provides a way to standardize the solidcontent of compositions according to the invention and to compare theantimicrobial activity of different aqueous extracts. For example, theamount of the anhydrous extract may be adjusted to a concentrationsufficient to inhibit growth of a Gram-positive bacterium, such asCellulomonas strain B22 or other strains such as Bacillus sp. B22 (2014)incorporated by reference to hypertext transferprotocol://www.uniprot.org/taxonomy/1529852 (last accessed Jun. 7,2017).

In another embodiment, an original, concentrated, purified, or solidaqueous extract will be formulated to contain one or more otheringredients such as a carrier, excipient, or support such as thosedescribed herein. Nonlimiting examples of such embodiments include thosein the form of an aqueous solution, tincture, humectant, absorbent,desiccant, dry gel, or gel; in the form of an oil-in-water orwater-in-oil emulsion; in the form of a serum, cream, or a lotion; inthe form of a spray or foam; in the form of a bandage, hydrogel patch,compress, dressing, bed linens, tamponade, tampon, gauze, gauze sponge,medical dressing, diaper or wipe. These products may comprise natural orsynthetic components, such as woven or non-woven fibers, textiles orelastic components. Fibers can be customary synthetic or semi-syntheticfibers, such as polyesters, polyolefins and rayon, or customary naturalfibers, such as cotton. A textile material can comprise fibers ofcellulose materials (e.g. cotton), viscose, linen, flax, hemp, jute andother natural fiber materials. It can also comprise fibers of syntheticfibers such as polyesters, polyamides, polyurethanes, polynitrile, ABS,polyolefins (such as polypropylene) as well as copolymeric materials,such as elastomeric materials and thermoelastic polymers (TPEs).

Another embodiment of the invention is a method for inhibiting growth ofa Gram-positive bacterium or mycoplasma comprising contacting it with anaqueous extract of leaves of Eruca sativa (arugula). Generally, thismethod will be performed using an extract from which is formulated asdescribed herein, for example in the form of an aqueous solution, a gel,tincture, oil-in-water or water-in-oil emulsion, a cream, a lotion,ointment, paste or powder. Growth or viability of Gram-positive bacteriaand/or mycobacteria is inhibited by contact with the extract or with aformulation containing the extract. Advantageously, these formulationsmay contain 0.01, 0.05, 0.1, 0.2, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 5.0,10.0, 15.0, 20.0 or more wt % of the aqueous extract, wherein the wt %of the aqueous extract is based on weight of anhydrous solids in anaqueous extract of Eruca sativa leaves. This range includes allintermediate subranges and values. In one embodiment the amount ofaqueous Eruca sativa extract is at least 0.15 g/ml.

Another embodiment of the invention is a method for treating a wound orinjury (including dermatitis or inflammatory conditions some of whichare associated with bacterial infection) comprising contacting it with acomposition comprising an aqueous extract of leaves of Eruca sativa.Generally, this method will be performed using an extract from which isformulated as described herein, for example in the form of an aqueoussolution, a gel, tincture, oil-in-water or water-in-oil emulsion, acream, a lotion, ointment, paste or powder. Healing of the wound orinjury or dermatitis or inflammatory condition is promoted by contactwith the extract or with a formulation containing the extract.Advantageously, these formulations may contain 0.01, 0.05, 0.1, 0.2,0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0 or more wt % of theaqueous extract (or any intermediate value or endpoint), wherein the wt% of the aqueous extract is based on weight of anhydrous solids in anaqueous extract of Eruca sativa leaves.

This method may be used to treat wounds, such as punctures, lacerations,abrasions, bites (e.g., mosquito or other insect bites), acne,inflammations, or other lesions in or on the skin, hair, nails, ormucous membranes. Such wounds include pressure ulcers, diabetic ulcers(e.g., diabetic foot ulcers), venous ulcers, lower leg ulcer; bedsores,blisters, eschars/scabs, scalds and burns (first, second and thirddegree burns), chemical burns, thermal burns such as flame burns andflash burns, ultraviolet burns, contact burns, radiation burns,electrical burns, gangrene, skin tears or lacerations, such as thosemade by knives; abrasions; punctures such as made by nails, needles,wires, and bullets; incisions such as made by knives, nails, sharpglass, razors; amputations; post-operative infections; surgical wounds;spider (including brown recluse bites), scorpion, centipede, tick, fly,mosquito, bites or stings; failing or compromised skin/muscle grafts orflaps.

This method may also be used to treat wounds in other anatomicallocations to which the aqueous extract can be applied or administered,for example, to ulcers or lesions in the GI tract.

In other embodiments it may be used to treat damage associated withaging such as wrinkles, dry skin, age spots, sun damage (particularly UVradiation-induced oxidative stress), blemishes, hyperpigmented skin, agespots, increased skin thickness, loss of skin elasticity and collagencontent, dry skin, lentigines and melasmas. In such embodiments, theextract or a formulation containing it is generally contacted with thedamage skin or tissue.

In another embodiment an Eruca sativa extract can be used or applied asan anti-microbial agent, for example, for the control of cellulosedegradation by Cellulomonas uda. This microorganism causes significantcellulose degradation and disintegration of plant tissue. A waterextract of Eruca sativa can be used by itself or as a source ofantibacterial compounds against Cellulomonas uda. Such an extract orextract components may be used in agriculture as natural compounds tocontrol plant pathogenic bacteria and other microorganisms that cancause serious losses in important crops, vegetable and fruit plant. Itmay also be applied to seeds, grain, fruit, vegetable, or other edibleportions of a plant (or to a food in general) to inhibit growth ofbacteria, increase shelf life, or improve commercial appearance of afood product. Furthermore, these naturally sourced extracts can be usedto partially or fully replace synthetic pesticides (includingbacteriocidal and bacteriostatic agents) and reduce or eliminate thetoxic and other negative effects of synthetic agents. Such extracts inliquid or solid form may be applied by means known in the art includingby sprayers or dusters.

The following examples further describe and demonstrate embodimentswithin the scope of the present invention. The examples are given solelyfor the purpose of illustration and are not to be construed aslimitations of the invention. Many variations thereof are possiblewithout departing from the spirit and scope of the present invention.

Example 1 Preparation of Eruca sativa Plant Extract

Extraction is the crucial first step in the analysis of medicinalplants, because it is necessary to extract the desired chemicalcomponents from the plant materials for further separation andcharacterization. The basic operation generally includes pre-washing,drying of plant materials, or freeze drying, grinding to obtain ahomogenous sample and improve the kinetics of analytic extraction.

Air-dried Eruca sativa leaves were ground to a fine powder using agrinder and the resulted material (100 g) was extracted by macerationinto 600 ml of water at room temperature with occasional shaking.

After three days, the extract was filtered (45 mm) and was concentratedunder vacuum to produce a thick concentrated extract.

The concentrated extract was weighed and preserved in airtight bottlesat 4° C. until further use. The resulted residue (22 g) was suspended in100 ml of distilled water. These fractions were kept at 4° C. in thedark until use or further analysis.

Example 2 Preparation of Cold Water Aqueous Extract of Eruca sativaLeaves

100 grams of dried, powdered Eruca sativa leaves are suspended in 1liter of water at room temperature (25° C.) and stirred mechanically for12 hours. Solids are removed by centrifugation (4,000 g, 10 mins) andthe supernatant is collected. The resulting aqueous extract iscompletely dried in a rotary evaporator at 40° C. and the lyophilizedextract is stored at 4° C.

Example 3 Preparation of Hot Water Aqueous Extract of Eruca sativaLeaves

100 grams of dried, powdered Eruca sativa leaves are suspended in 1liter of water at room temperature (80° C.) and stirred mechanically for1 hour. Solids are removed by centrifugation (4,000 g, 10 mins) and thesupernatant is collected. The resulting aqueous extract is completelydried in a rotary evaporator at 40° C. and the lyophilized extract isstored at 4° C.

Example 4 Preparation of a Filtered Cold Water Aqueous Extract of Erucasativa Leaves

100 gr of dried Eruca sativa leaves are mashed by pressing and mixedwith 1,000 ml of cold water for 12 hours at 25° C. Undissolvedcomponents are removed from the resulting suspension by centrifugation(4,000 g, 10 mins) and then from the supernatant by filtration through a0.45 micron filter. The resulting aqueous extract is completely dried ina rotary evaporator at 40° C. and the lyophilized extract is stored at4° C.

Example 5 Preparation of a Filtered Hot Water Aqueous Extract of Erucasativa Leaves

100 gr of dried Eruca sativa leaves are mashed by pressing and mixedwith 1,000 ml of water and mechanically stirred in boiling water for 30mins at 80° C. Undissolved components are removed from the resultingsuspension by centrifugation (4,000 g, 10 mins) and then from thesupernatant by filtration through a 0.45 micron filter. The resultingaqueous extract is completely dried in a rotary evaporator at 40° C. andthe lyophilized extract is stored at 4° C.

Example 6 Assessment in Liquid Culture of Antimicrobial Activity ofAqueous Extract of Eruca sativa Leaves

Water extracts of Eruca sativa as described in Examples 1-5 arecontacted with Gram-positive and Gram-negative bacteria. The effect ofEruca sativa water extracts on the growth of Gram-positive test strainB22 is determined by measuring the optical density (OD) of treated anduntreated bacterium and counting the number of viable cells (cfu/mL).Tested bacteria include those described below as well as Gram-negativestrains.

Bacteria Effect Rhodococcus a plant pathogen, causes leafy gall spdisease in both angiosperm and gymnosperm plants Gordonia emerging humanpathogen that sputi causes a variety of infections in bothimmunocompromised and immunocompetent hosts, Cutaneous and respiratoryinfections, otitis externa, osteitis, and arthritis have reportedlyoccurred only in immunocompetent patients. Cellulomonas cellulosedegradation and uda disintegration of plant tissue Bacillus soft rot onapple and pear fruits altitudinis

Minimum inhibitory concentrations (MIC) of the extract of Example 1 weredetermined according to Eloff (1998) in sterile 96-well microplates witha final volume of 100 ml in each microplate well. A stock solution ofthe water extract (250 mg/ml) was prepared. Thereafter, a two-foldserial dilution of the extract was prepared in the microplate wells overthe range 0.02-0.25 mg/ml. To each test well, 5 ml of cell suspensionwas added to reach a final inoculum concentration of 10⁶ cfu/ml. Theplates were then covered with sterile plate covers and incubated at 30 Cfor 48 h. The MIC was defined as the lowest concentration of the extractat which the microorganism does not demonstrate visible growth afterincubation. The lowest concentration that yielded no growth after thissub-culturing was taken as the MBC, indicating that >99.9% of theoriginal inoculum was killed. The determination of MIC and MBC valueswas properly replicated three times.

Antimicrobial activities of the Eruca sativa extract were evaluated bymeans of agar-well diffusion assay according to the method of Andrews(2005) with minor modifications. Fifteen milliliters of the molten agar(45° C.) were poured into sterile petri dishes (Ø90 mm). Cellsuspensions were prepared and 100 ml was inoculated onto the surface ofagar plates. Thereafter, wells with 6 mm in diameter were punched in theinoculated agar medium with sterilized Pasteur pipettes and the extractswere added to each well. Negative controls consisting of organic solventwere used to dissolve the plant extracts. The plate was allowed to standfor 2 h at 4° C. to permit the diffusion of the extracts followed byincubation at 30° C. for 48 h. The antibacterial activity was evaluatedby measuring the zones of inhibition (clear zone around the well)against the tested microorganisms. All tests were repeated three times.

In this assay, only Gram-positive bacteria were susceptible to theextract and the Gram-negative bacteria were not inhibited at the sameconcentrations.

Different concentrations of the water extracts are added to differentsamples of culture media. The results of these experiment show thataqueous extracts of Eruca sativa leaves inhibited the growth of B22strain at 0.15 g/mL (dry weight of extract/volume).

While not being bound to any particular explanation, this resistance isattributed to differences in cell wall structure between Gram-positiveand Gram-negative bacteria. Gram-negative bacteria have a permeabilitybarrier comprised of a thin lipopolysaccharide exterior membrane whichcould restrict the penetration of the extruding the plant extract. Incontrast, Gram-positive bacteria have a mesh-like peptidoglycan layerwhich may be more accessible to permeation by the Eruca sativa extract.

Example 7 Assessment on Agar Plates of Antimicrobial Activity of AqueousExtract of Eruca sativa Leaves

Gram-positive or Gram-negative bacteria are plated on to agar platescontaining a medium supporting their growth. Thin permeable wafers/discsare infused with control solution (water) or with various concentrationsof aqueous extracts of Eruca sativa as described in Examples 1-5. Plateswith wafers/discs are incubated at 37° C. overnight. The growth ofGram-positive bacteria is inhibited by the Eruca sativa leaf extracts ata concentration of 0.15 g/mL (dry weight of extract/volume) as shown bythe appearance of a zone of inhibition where growth of Gram positivebacteria is not observed. In contrast, the zone of inhibition for Gramnegative bacteria is while the growth of Gram-negative bacteria at thesame concentrations is not substantially inhibited as shown by theabsence of, or substantially smaller zones of inhibition.

Terminology used in this disclosure is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. For example, as used herein, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

It will be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements, and/or components, but donot preclude the presence or addition of one or more other features,steps, operations, elements, components, and/or groups thereof. Forexample, the term “comprising” will be understood to imply the inclusionof any stated elements or steps but not the exclusion of any otherelements or steps. Likewise, the terms “include”, “includes” and“including”, unless the context requires otherwise, do not excludeunrecited elements or steps.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

Links are disabled by insertion of a space or underlined space before“www” and may be reactivated by removal of the space.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/−0.1% of the stated value (orrange of values), +/−1% of the stated value (or range of values), +/−2%of the stated value (or range of values), +/−5% of the stated value (orrange of values), +/−10% of the stated value (or range of values),+/−15% of the stated value (or range of values), +/−20% of the statedvalue (or range of values), etc. Any numerical range recited herein isintended to include all subranges and values subsumed therein.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of different embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application isspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears. However, no admission is made withregard to the accuracy of the reference teachings or that thesereferences are applicable prior art.

The invention claimed is:
 1. A method for killing or inhibiting thegrowth of a Gram positive bacterium while preventing desiccation of awound, comprising: preparing by: grinding a sample consisting of driedleaves of a Eruca sativa plant, macerating the ground leaves of theEruca sativa plant with an extractant consisting of water to extract theleaves of the Eruca sativa plant for a period of days at a temperatureof about 25° C. to extract water-soluble components from the leaves ofthe Eruca sativa plant then filtering to form a first extract,concentrating the first extract under vacuum to produce the aqueousEruca sativa extract in the form of a thick concentrated extract liquid,then dispensing an aqueous composition comprising the thick concentratedextract liquid from a dispenser in the form of a foam, contacting theGram positive bacterium with the foam to inhibit the growth of the Grampositive bacterium while preventing desiccation of the wound, whereinthe aqueous composition inhibits the growth or kills Cellulomonas strainB22.
 2. The method of claim 1 that is a method for killing Gram-positivebacteria.
 3. The method of claim 1 that is a method that inhibits growthof the Gram positive bacteria while preventing desiccation of a wound,wherein said contacting the Gram positive bacterium with the foam toinhibits the growth of the Gram positive bacterium occurs whilepreventing desiccation of the wound.
 4. The method of claim 1, whereinthe composition is formulated as a bandage, hydrogel patch, compress, ordressing.
 5. The method of claim 1, wherein the wound comprises apuncture, laceration, abrasion, or bite.
 6. The method of claim 1,wherein the wound comprises a burn.
 7. The method of claim 1, whereinsaid composition comprises no more than 0.5 wt. % of the aqueous extractas measured after removal of water.